ELISA has long been used to detect and measure antibody (or antigen) levels. Most commonly, ELISA is used as a serological assay, but it is also used to study the immunochemical properties of antigens or antibodies, and has frequently found application in, for example, the evaluation and characterization of immune responses, to investigate antibody production by cell cultures, and in hybridoma technology.
The ELISA assay is simple to use, sensitive and relatively quick, but it is only able to simply measure the presence of the target antibody in the sample; it cannot distinguish between on-going antibody synthesis in response to the antigen, and antibodies already present from past infection, or by passive transfer. Whilst in some cases, it may suffice simply to obtain information concerning the presence of antibody, it is in other cases desirable to be able to determine whether or not the detected target antibodies are acutely synthesized by the lymphocytes at the time of testing, for example during a vaccination course, or in the diagnosis of infection in infants, to distinguish from passively transferred maternal antibodies. This cannot be achieved in a classical ELISA method.
Other methods have therefore been developed, which enable on-going antibody synthesis to be detected. Particular mention may be made in this regard of the enzyme-linked immunospot (ELISPOT) assay (also known as spot ELISA or ELISA-plaque assay), as reviewed for example by Czerkinsky et al. in ELISA and other Solid Phase Immunoassays, Ed. D. M. Kemeny and S. J. Challacombe, 1988, Chapter 10, 217-239. This technique, based on the ELISA method, enables the enumeration of lymphocytes secreting antibodies against one or more target antigens. Basically, the ELISPOT is a variant of the ELISA method, whereby antibody secreting cells (ASC) may be revealed by culturing lymphocytes in specially modified ELISA wells coated with the target antigen, and by replacing the standard ELISA reagents with enzyme-substrate complexes that yield a colored precipitate (spots), adjacent to the secreting cell. Spots can then be counted to give a measure of the number of antibody-producing cells. Protein synthesis inhibitors may be included in the culture medium, to confirm that the spots detected are due to de novo antibody synthesis, during the in vitro incubation period.
Whilst the ELISPOT technique has proved very useful in studying the dynamics of humoral immune responses, and has been used to detect spontaneous ASC that appear transiently in the peripheral circulation of immunized subjects, certain features of the method place constraints on its use in a clinical diagnostic setting. Firstly, since for each sample individual spots need to be counted which can be time consuming and laborious, the method is not particularly suited to the analysis of large numbers of samples, such as occurs in a clinical diagnostic laboratory. Secondly, only the number of cells secreting antibody in each sample is enumerated and generally speaking, this requires reasonably large sample volumes, e.g., several milliliters. ELISPOT plates are also expensive and the assay is not readily amenable to automation.
WO 96/26443 describes the use of a modified ELISA test which may be used to detect on-going antibody synthesis. In this assay lymphocytes are cultured after isolation and the levels of antibody produced during that culture period is determined. This technique thus necessarily requires incubation of the lymphocytes of the test sample at about 37° C. in order to allow measurement of the antibodies secreted during incubation. The average incubation period is 2-5 hours, which represents a significant limitation on the speed of performing the assay. Incubation also requires the provision of suitable equipment at the site of testing so that it may be carried out immediately prior to the assay procedure. This generally means that assay of the lymphocytes needs to be carried out shortly after a sample has been taken from a subject, because storage of samples, e.g., by freezing is not acceptable due to the resulting decrease in cell viability. Purified cells can only be maintained viable for relatively short periods of time by storage on ice at 0° C. or less favorably at 4° C.
It will be seen therefore, that despite advances in antibody detection techniques, there remains a need for a further improved assay which is simple, quick and cost effective to perform, which reliably enables precise quantification of spontaneously secreted antibody, which is able to distinguish the antibody synthesis, which may be performed on samples, e.g., blood samples, for diagnostic purposes. The present invention addresses this need.
In the above described technique, a period of culture was used in line with current thinking at the time on the assumption that this was necessary in order to obtain sufficient antibody titre for obtaining assay results for immunodiagnostic purposes.
It has been established in the literature that the biosynthesis of antibodies (immunoglobulins) takes place in B lymphocytes (see: Roitt I, Brostoff J, Male D. “Immunology”, 4th Edition published by Mosby, London 1996, 6.12-6.13) whereafter they are secreted into the blood stream to fight infection. When secreted by antibody-secreting cells (ASC) the molecules are fully assembled (e.g. for an IgG molecule two heavy chains and two light chains joined by disulphide bonds) and glycosylated. The rate-limiting step in the biosynthesis of antibodies has been claimed to be the intracellular transport and glycosylation via the endoplasmic reticulum and Golgi complex (which takes 1 hour or more), whereas the biosynthesis of the various heavy and light chains of the immunoglobulins only needs minutes for completion. In total, the process of synthesis and secretion is estimated to take in the order of 2 hours (Melchers, 1971, Histochemical, J., 3, p 389-397). As a consequence of the rapid secretion of immunoglobulins from cells, it has never before been appreciated that functional antibodies, or even partially synthesized antibodies (e.g. preglycosylation) could be present within the lymphocyte cell in any significant amount. Furthermore it was not appreciated that disruption of lymphocytes in a sample could yield sufficient quantities of “newly synthesized antibodies” to allow detection for immunodiagnostic purposes.